MX2008001978A - Vaccination against dengue virus infection - Google Patents

Abstract

This invention relates to methods and kits for use in vaccination against dengue virus infection.

Description

VACCINATION AGAINST INFECTION WITH DENGUE VIRUS Background of the Invention This invention relates to vaccination against dengue virus infection. Dengue, a disease caused by four different species of the dengue virus (called serotypes 1-4), is the most important vector-borne disease of humanity. Approximately 100 million people are affected by dengue virus annually in tropical and subtropical regions of the world (Halstead, "Epidemiology of Dengue and Dengue Hemorrhagic Fever," CABI Publ., New York, pp. 23-44, 1997; Gubler, " Dengue and Dengue Hemorrhagic Fever ", CABI Publ., New York, pp. 1-22, 1997). A severe and potentially lethal form of disease caused by dengue virus infection, dengue hemorrhagic fever (DHF), is increasing in geographic distribution and incidence. These events have led to intense efforts to build safe and effective vaccines against dengue but, despite many efforts, covering more than 50 years, no commercially available vaccine against dengue has been developed. The development of a vaccine against dengue is thus considered a high priority by the World Health Organization (Chambers et al., Vaccine 15: 1494-1502, 1997).

The pathogenesis of DHF drives the design of dengue vaccines. DHF is an immunopathological disease, which occurs mainly in individuals who have sustained a previous infection with a dengue serotype and then are exposed to a second different serotype (heterologous). Infection with any one of the four dengue serotypes provides durable immunity against that homologous serotype, based on neutralizing antibodies. However, immunity against other heterologous dengue serotypes after infection with a dengue serotype is of short duration, if it occurs at all (Sabin, Am. J. Trop. Med. Hyg. 1: 30-50, 1952). Typically, after a few weeks or months, only binding and non-neutralizing antibodies to heterologous serotypes are present. These ligation but non-neutralizing antibodies can increase subsequent infection with a heterologous serotype of the dengue virus, increasing the risk of severe disease (Rothman et al., Virology 257: 1-6, 1999). Given the immunopathogenesis of DHF, a successful vaccine against dengue should be safe and induce long-lasting, cross-neutral antibody responses against all 4 serotypes of the dengue virus simultaneously, so that titres do not fall to levels that leave a subject not protected against future infection. Historically, empirical efforts to develop live, attenuated vaccine candidates have shown that it is difficult to achieve a balance between sufficient attenuation (safety) and immunogenicity of candidate vaccine viruses. It has also been difficult to combine vaccine strains representing all four serotypes in an effective tetravalent mixture and a multiple dose program was necessary to achieve seroconversion against all serotypes, with the undesirable effect of providing spaces in the immunization program where subjects could become sensitized with immunopathological events. In fact, attempts to immunize with live, monovalent dengue vaccine mixtures, demonstrated significant interactions between the four strains of virus and have resulted in viral interference effects (reviewed in Saluzzo, Adv. Res. Virus 61: 420-444 , 2003). Vaccines based on chimeric flaviviruses genetically engineered against dengue virus have been developed, in which two sequences (ie, sequences encoding the pre-membrane (prM) and capsule (E)) proteins of dengue 1 serotypes, 2, 3, or 4 are inserted into a full-length infectious clone of yellow fever virus 17D, instead of the sequences encoding the corresponding yellow fever virus proteins (see, e.g., Guirakhoo et al., J. Virol 75: 7290-7304, 2001; Guirakhoo et al., Virology 298: 146-159, 2002). These viruses are highly effective in inducing immune responses when injected into monkeys. However, preliminary data also showed some effects of viral interference in humans, which can limit immunization against all four serotypes after a dose of dengue vaccine. The present inventors have found a new and safe method of immunization against dengue diseases, which allows induction of long-lasting, cross-neutral antibody response against dengue serotypes 1-4, while avoiding the need for a vaccination program against multiple dose dengue and the potential risk associated with an unbalanced primary immune response. The method of the present invention, which uses an immunization regimen comprising the administration of a first yellow fever vaccine followed by the administration of a dengue vaccine based on chimeric flavivirus, allows the induction of a cross neutralization immune response against dengue viruses, which has the advantages of appearing early (within 30 days) after the administration of the dengue vaccine, being long-lasting, and being cross-reactive against the four serotypes. Moreover, the method of the present invention has the additional benefit of inducing a protective immune response against yellow fever. Price et al. (Am. J. "Epid.88: 392-397, 1968) previously describes a method for sequential flavivirus immunization comprising a series of three immunizations with dengue type 2 and two heterologous viruses (yellow fever and Japanese encephalitis). Moreover, unlike the present invention, the sequence of yellow fever followed by dengue 2, without the addition of Japanese encephalitis immunization, failed to confer cross-protection immunity. Scott et al (J. Infecí Dis 148: 1055-1060, 1983), showed that subjects who were previously immunized with yellow fever and subsequently inoculated with a live, attenuated type 2 dengue vaccine have improved immune responses against dengue type 2, which are also more durable (lasting 3 years) than in subjects without prior yellow fever immunity. The improved response may have been due to increasing antibodies (binding, non-neutralizing) produced to the dengue virus type 2 by vaccination of preceding yellow fever (Eckels et al, J. "Immunol. 135 (6): 4201-4203, 1985) However, Scott and colleagues did not show that yellow fever vaccines followed by dengue 2 produced a long-term immune response to the other three dengue serotypes (types 1, 3, or 4). , the sequence of yellow fever followed by dengue type 2 was not shown to produce a wide response by the neutralization test, which is the only test that predicts protective immunity.In a recent article, Kanesa-thasan et al. (Am. Trop.Med. Hyg. 69 (Suppl 6): 32-38, 2003) discovered promulgated heterologous responses and anti-dengue antibody titers in subjects remotely vaccinated with yellow fever following vaccination with attenuated dengue vaccines. These short-term antibody responses (at day 30) were demonstrated with antibody assays including neutralization, but the authors concluded that evidence for protection against subsequent dengue infection was inconclusive. In contrast to the present invention, the authors could not conclusively demonstrate the previous time or receipt of yellow fever vaccination, long-term broad neutralizing antibody responses, or provide evidence for cross-reactive T-cell responses to dengue. The present inventors demonstrate for the first time that induction of cross-neutralization immunity against multiple dengue serotypes in humans can in fact be conferred by sequential administration of chimeric yellow fever and dengue viruses. SUMMARY OF THE INVENTION The present invention provides a method for inducing a long-term, cross-neutral immune response to the dengue virus in a patient, comprising administering to the patient: (i) a dose of a yellow fever virus vaccine, and (ii) a dose of chimeric flavivirus vaccine comprising at least one chimeric flavivirus comprising a yellow fever virus backbone in which the sequence which encodes envelope protein of the yellow fever virus has been replaced with a sequence encoding the envelope protein of a dengue virus, where the chimeric flavivirus vaccine is administered at least 30 days and up to 10 years after administration of the yellow fever vaccine. In one example, the dengue capsule sequence is a revolving sequence. According to one embodiment, the chimeric flavivirus comprises a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with sequences encoding the membrane proteins and of a dengue virus capsule. In one example, either or both of these dengue sequences are scrambled sequences. According to a particular embodiment, the chimeric flavivirus vaccine is administered to the patient 30, 60, or 90 days after administration of the yellow fever vaccine. According to a particular embodiment, the chimeric flavivirus used in the dengue vaccine of the invention is composed of a skeleton of yellow fever virus 17D (YF17D). According to another embodiment, the yellow fever virus vaccine used in the method of the invention comprises a YF17D strain.

According to another embodiment, the chimeric flavivirus vaccine used in the method of the present invention comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the virus of Yellow fever have been replaced with sequences that encode the membrane and capsule proteins of a serotype 1 dengue virus. According to another embodiment, the chimeric flavivirus vaccine used in the method of the invention comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the fever virus have been replaced with sequences that encode the membrane proteins and capsule of a virus of serotype 2 of dengue. According to another embodiment, the chimeric flavivirus vaccine used in the method of the invention comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the membrane proteins and fever virus capsule They have been replaced with sequences that encode the membrane and capsule proteins of a virus of serotype 3 of dengue. According to another embodiment, the chimeric flavivirus vaccine used in the method of the invention comprises a chimeric flavivirus comprising a virus backbone. yellow fever in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with sequences that encode the membrane and capsid proteins of a serotype 4 virus of dengue. According to a particular embodiment, the chimeric flavivirus vaccine used in the method of the present invention is a monovalent vaccine or a tetravalent vaccine. According to another embodiment, the method of the invention further comprises administration of a booster dose of the chimeric flavivirus vaccine defined above, 6 months to 10 years after the first dose of the chimeric flavivirus vaccine. According to another aspect, the present invention relates to a kit comprising: (i) a yellow fever virus vaccine, and (ii) a chimeric flavivirus vaccine comprising at least one chimeric flavivirus comprising a skeleton of yellow fever virus in which the sequence encoding the capsule protein of the yellow fever virus has been replaced with the sequence encoding the capsid protein of a dengue virus. In one example, the dengue capsule sequence is a revolving sequence. According to one embodiment, the chimeric flavivirus comprises a skeleton of yellow fever virus in which the sequences encoding the membrane proteins and of yellow fever virus capsule have been replaced with sequences that encode the membrane proteins and capsule of a dengue virus. In one example, either or both of these dengue sequences are scrambled sequences. According to one embodiment of the kit of the invention, the yellow fever virus vaccine comprises a YF17D strain, wherein YF17D comprises a number of sub-strains used for vaccination against yellow fever (including 17D-204, 17D-213 and 17DD). According to another embodiment, the chimeric flavivirus is composed of a YF17D virus skeleton. According to another embodiment of the kit of the invention, the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a YF17D virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with sequences encoding the membrane and capsule proteins of a serotype 1 dengue virus. According to another embodiment of the kit of the invention, the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the YF17D virus have been replaced with sequences that encode the membrane proteins and capsule of a virus of serotype 2 of dengue.

According to another embodiment of the kit of the invention, the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the YF17D virus have been replaced with sequences that encode the membrane proteins and capsule of a virus of serotype 3 of dengue. According to another embodiment of the kit of the invention, the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a YF17D virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with sequences encoding the membrane and capsule proteins of a dengue serotype 4 virus. According to another embodiment, the kit as defined above also comprises at least one booster dose of a chimeric flavivirus vaccine as defined above. According to another embodiment, the invention relates to the use of the viruses mentioned above and elsewhere in the present in the prevention and treatment of dengue virus infection, as well as the use of these viruses in the preparation of drugs. for this purpose. Definitions "Immune neutralization cross response" is understands a specific immune response comprising neutralization antibodies against multiple different dengue serotypes (up to 4). Induction of a cross neutralization immune response can be easily determined by a reference plate neutralization assay (PRNT50). For example, induction of a cross neutralization immune response can be determined by one of the PRNT50 assays as described in example 1. A serum sample is considered positive for the presence of cross-neutralizing antibodies when the neutralizing antibody titer as well determined is at least greater than or equal to 1:10 in at least one of these trials. By "long-term immune response" is meant a positive cross-neutralization immune response as defined above, which can be detected in human serum for at least 6 months, advantageously, at least 12 months after the administration of a vaccine of chimeric flavivirus as defined below. "Patient" means individuals susceptible to yellow fever, including adults and children. Individuals "susceptible to yellow fever" means individuals without documented vaccination against yellow fever for more than 10 years and / or without infection of certified yellow fever virus for more than 10 years. By "individuals immune to yellow fever" is meant, within the framework of the present invention, individuals with a documented vaccination against yellow fever and / or with a certified yellow fever virus infection that has occurred 10 years ago or less, eg, 5 years or less, v.gr ., 4, 3, 2, or 1 year ago, or even 6, 5, 4, 3, or 2 months ago, and in some cases more than 30 days ago. By "chimeric flavivirus" is meant a chimeric flavivirus composed of a yellow fever virus skeleton in which the sequence encoding the capsule protein of the yellow fever virus has been replaced with the sequence encoding the capsule protein of a virus of dengue Advantageously, a chimeric flavivirus is composed of a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with the sequences encoding the membrane and capsule proteins of a virus of dengue The yellow fever skeleton can advantageously be from a vaccine strain, such as YF17D or YF17DD. These chimeric flaviviruses are defined in more detail below and are named YF / dengue-N, with N identifying the dengue serotype. By "chimeric flavivirus vaccine" is meant an immunogenic composition comprising an immuno-effective amount of at least one chimeric flavivirus as defined above and a pharmaceutically acceptable excipient. The chimeric flavivirus vaccine is said to be "monovalent" when the vaccine comprises chimeric flavivirus that expresses the protein of a dengue serotype. Examples of monovalent vaccines are vaccines comprising YF / dengue-1, YF / dengue-2, YF / dengue-3, or YF / dengue-4, advantageously YF / dengue-2. The chimeric flavivirus vaccine is said to be "bivalent" when the vaccine comprises chimeric flaviviruses that express proteins from two different dengue serotypes. Examples of bivalent vaccines are vaccines comprising YF / dengue-2 and YF / dengue-4, or YF / dengue-2 and YF / dengue-3, or YF / dengue-2 and YF / dengue-1. The chimeric flavivirus vaccine is said to be "trivalent" when the vaccine comprises chimeric flaviviruses that express proteins from three different dengue serotypes. Examples of trivalent vaccines are vaccines comprising YF / dengue-2, YF / dengue-1, and YF / dengue-4, or YF / dengue-2, YF / dengue-3, and YF / dengue-4. The chimeric flavivirus vaccine is said to be "tetravalent" when the vaccine comprises chimeric flaviviruses that express proteins from four different dengue serotypes. An example of a tetravalent vaccine is a vaccine that includes YF / dengue-1, YF / dengue-2, YF / dengue-3, and YF / dengue-4. By "immuno-effective amount of a chimeric flavivirus" is meant an amount of a chimeric flavivirus capable of inducing, after administration in a subject immune to yellow fever, a cross-neutralized immune response as defined above. Typically, an immunoefectivating amount of a chimeric flavivirus is comprised between 102 and 107, e.g., between 103 and 106, such as an amount of 104, 105, or 106, infectious units (e.g., forming units). of plaques or infectious doses in tissue culture) by serotype, per dose. A central advantage of the method of the present invention is the ability to induce neutralizing antibodies against all four dengue serotypes rapidly and simultaneously, thereby protecting against dengue fever and thus avoiding the potential associated risks of developing dengue haemorrhagic fever in natural exposure subsequent to dengue infection. Neutralizing antibodies directed against the dengue capsule protein are considered the main mediator of protective immunity against infection, therefore the demonstration of neutralizing antibodies is considered as a relevant substitute for a neutralizing immunity in patients. Other features and advantages of the invention will be apparent from the following detailed description, the claims, and the drawings. Brief Description of the Drawings Figure 1 is a graph showing responses of IFN? to vaccination (Day 31 of study minus Day 1). The two doses of ChimeriVax-Den2 gave equivalent T cell responses. The response was not inhibited in subjects previously vaccinated with a yellow fever virus vaccine.

Detailed Description The invention provides a method for inducing long-term, cross-neutralization immunity in a patient for all four dengue serotypes (1-4) using a simple two-step procedure. The target population is thus composed especially of the following patients at risk of dengue infection: travelers abroad, expatriates and military personnel, as well as inhabitants of regions in which dengue is endemic. In this method, a patient is first immunized with a dose (preferably a dose, but possibly more than one dose (eg, 2 or 3 doses)) of a yellow fever virus vaccine (e.g. a live attenuated vaccine, commercially available, see below). After an appropriate time interval of at least 30 days, which allows in particular for the resting state of the innate immune response induced by the yellow fever virus vaccine, the second step in the method is carried out, which involves the administration of a dose of a chimeric flavivirus vaccine comprising one or more live attenuated chimeric viruses, each comprising a yellow fever virus skeleton in which one or more sequences encoding structural proteins (e.g. pre-membrane and capsule proteins) have been replaced with the sequences encoding the corresponding proteins of a dengue virus (e.g., dengue 1, 2, 3, or 4). The present inventors have shown that this immunization sequence produces high neutralizing antibody titers against all four dengue serotypes. These antibodies persist at high levels for 6 months and even 12 months after the administration of the dengue vaccine, indicating that immunity against broad dengue was long-term. Since the initial immunizing / primary agent (yellow fever vaccine) is incapable of sensitizing the subject to DHF, there is no danger that the primary inoculation, first, leaves the subject vulnerable to this disease if the second injection is delayed or not taken finished. These results were unexpected, such as a sequential infection with two serotypes of the dengue virus, which are much more closely related to each other based on genome sequence and antigenic relationships than yellow fever is related to dengue. they induce solid protection or broad cross neutralization antibody responses against infection with the two remaining dengue serotypes. Further demonstration of the unexpected nature of the sequential vaccination method of the invention was provided by a response examination of the yellow fever antibody following the second step (inoculation of the chimeric dengue virus). The method of the invention is further described, as follows. Yellow Fever Virus Vaccines As mentioned above, the first step of the method of the invention involves administration to a patient of a dose of a yellow fever virus vaccine. Examples of such vaccines that can be used in the invention include live attenuated vaccines, such as those derived from strain YF17D, which was originally obtained by attenuation of the wild-type Asibi strain (Smithburn et al., "Yellow Fever Vaccination", World Health Organization, pp. 238, 1956, Freestone, in Plotkin et al. (Editors), Vaccines, 2nd edition, WB Saunders, Philadelphia, United States, 1995). An example of a YF17D strain from which vaccines that can be used in the invention can be derived is YF17D-204 (YF-VAX, Sanofi-Pasteur, Swiftwater, Pennsylvania, United States, Stamaril, Sanofi-Pasteur, Marcy- L 'Etoile, France, ARILVAX, Chiron, Speke, Liverpool, United Kingdom, FLAVIMUN, Bern Biotech, Bern, Switzerland, YF17D-204 France (X15067, X15062), YF17D-204, 234 US (Rice et al., Science 229: 726-733, 1985)), while other examples of such strains that can be used are closely related to strain YF17DD (GenBank Accession No. 17066), YF17D-213 (GenBank Accession No. U17067), and 17DD strains of yellow fever virus described by Galler et al., Vaccines 16 (9/10): 1024-1028, 1998. In addition to these strains, any other strain of yellow fever virus vaccine found to be acceptably attenuated in humans, such as human patients, can be used in the invention. The yellow fever virus vaccines used in the invention can be obtained from commercial sources (see above) or can be prepared using methods that are well known in the art. In one example of such methods, chick embryos are inoculated with virus at a fixed step level, and then virus isolated from centrifuged homogenate supernatants is freeze-dried. In other methods, the yellow fever strain is grown in cultured chicken embryo fibroblasts (see, v.gr, Freiré et al., Vaccine 23 (19) = 2501-2512, 2005) or other cells grown for vaccine manufacture. virals such as Vero cells. Yellow fever virus vaccines are usually stored in lyophilized form prior to use. When needed for administration, the vaccines are reconstituted in an aqueous solution (typically, about 0.5 mL), such as a 0.4% sodium chloride solution, and then administered by subcutaneous injection into, e.g., the deltoid muscle. Other modes of administration determined as being appropriate by those skilled in the art (e.g., intramuscular or intradermal injection, or percutaneous administration using methods that deliver viruses to the surface layers of the skin) can also be used. The vaccine can be administered in doses varying from, for example, 2-5 (e.g., 3 or 4) log10 plaque forming units (PFU) per dose. All commercially available vaccines are used according to the manufacturer's recommendations. In one embodiment, the first step of the method of the invention consists in the administration of a dose of Stamaril or a dose of YF-VAX. The method of the present invention can also be adapted for use with patients immune to yellow fever. In such a case, the method comprises only the second step involving the administration of a dose of a chimeric flavivirus vaccine as defined below. The said method is also included within the scope of the present invention. Chimeric Flavivirus Vaccines The second step of the immunization method according to the invention comprises administration of a dose of a chimeric flavivirus vaccine as defined above. For reasons of clarity, in the following description, the invention is only defined in relation to the use of chimeric flaviviruses in which the chimeric flavivirus is composed of a yellow fever virus skeleton in which the sequences encoding the membrane proteins and capsule of the yellow fever virus have been replaced with the sequences that encode the membrane and capsule proteins of a dengue virus. The invention also includes the use of other chimeras, such as chimeras in which only one protein (e.g., capsule protein) of a yellow fever vaccine strain has been replaced, or chimeras in which all three Structural proteins have been replaced. Chimeric viruses that can be used in the present invention include those based on the vaccine strain of human yellow fever, YF17D (e.g., YF17D-204, YF17D-213, or YF17DD), as described above. In these viruses, the pre-membrane and capsule proteins of the yellow fever virus are replaced with the pre-membrane and capsule proteins of a dengue virus (serotype 1, 2, 3, or 4). In one embodiment of the present invention, the chimeric viruses are composed of a YF17D-204 skeleton in which the sequences encoding the pre-membrane and capsule proteins of the yellow fever virus are replaced with the sequences encoding the proteins of pre-membrane and capsule of serotypes 1, 2, 3, and / or 4 of wild-type dengue, eg, with the sequences that encode pre-membrane and capsule proteins of dengue virus 1 PUO-359 , dengue virus 2 PUO-218, dengue virus 3 PaH-881/88, or dengue virus 4 1228. Details of the construction of these constructs of chimeric and related viruses are provided, for example, in the following publications : WO 98/37911; WO 01/39802; Chambers et al., J. Virol. 73: 3095-3101, 1999; WO 03/103571; WO 2004/045529; US Patent 6,696,281; US patent 6,184,024; US patent 6,676,936; US Patent 6,497,884; Guirakhoo et al., J. Virology 75: 7290-7304, 2001; Guirakhoo et al., Virology 298: 146-159, 2002; and Caufour et al., Virus Res. 79 (1-2): 1-14, 2001. As a specific example of a chimeric flavivirus that can be used in the invention, note is made of the following chimeric flavivirus, which was deposited in the American Type Culture Collection.

(American Type Culture Collection, ATCC) in Manassas, Virginia, United States, under the terms of the Budapest Treaty and granted a deposit date of January 6, 1998: Virus Yellow Fever 17D / Dengue Type 2 Chimeric (YF / DEN-2, Accession number ATCC VR-2593). The chimeric flaviviruses used in the methods of the invention can optionally include attenuating mutations in dengue virus sequences. For example, dengue sequences may include an amino acid deletion or substitution of capsule 204 (dengue serotypes 1, 2, and 4) or 202 (dengue serotype 3), which is lysine in wild type viruses. In one example of such a substitution, the lysine in this position is replaced with arginine. In other examples, one or more other amino acids in the amino acid region 200-208 (or combinations of these amino acids) is mutated, with specific examples including the following: position 202 (K) of dengue 1; position 202 (E) of dengue 2; position 200 of dengue 3 (K); and positions 200 (K), 202 (K), and 203 (K) of dengue 4. These residues can be substituted with, for example, arginine. These mutations are described in detail in WO 03/103471, the content of which is incorporated herein by reference. In addition to the chimeras described above, other chimeras that contain structural proteins including epitopes of more than one serotype of dengue virus (2, 3, or 4) can be used in the invention. In an example, chimeras can to be made using stir technology, which involves cycles of fragmentation, assembly, and selection of sequences that are being disrupted (see, e.g., Locher et al., DNA Cell Biol. 24 (4): 256-263, 2005) . Thus, in the present case, sequences encoding capsule and / or pre-membrane proteins from a desired subset of dengue serotypes (or all dengue serotypes) can be processed in this manner to generate capsule sequences and / or pre-membrane revolves, which are then used to replace the corresponding sequences of a yellow fever virus backbone as described herein (e.g., YF17D). Such a chimeric YF / Denl-4 revolver (assuming that the scrambled sequences include epitopes of all four serotypes) can be produced by, for example, transfection of Vero cells with chimeric RNA transcripts and recovery of live virus from the supernatant as previously described (Guirakhoo et al, J. Virol. 75 (16): 7290-7304, 2001) and mentioned in other points herein. These scrambled chimeras can be used in the invention in vaccination regimens involving administration of the chimera revolving following yellow fever vaccination (e.g., YF17D), or in any of the combination methods described elsewhere herein. The chimeric viruses described above can be made using standard methods in the art. For example, an RNA molecule corresponding to the genome of a virus can enter primary cells, chicken embryos, or diploid cell lines, from which (or the supernatants of which) progeny virus can then be purified. Other methods that can be used to produce the viruses employ heteroploid cells, such as Vero cells (Yasumura et al., Nihon Rinsho 21: 1201-1215, 1963). In an example of such methods, a nucleic acid molecule (e.g., an RNA molecule) corresponding to the genome of a virus is introduced into the heteroploid cells, virus is harvested from the medium in which the cells have been harvested. When cultured, harvested viruses are treated with a nuclease (e.g., an endonuclease that degrades both DNA and RNA, such as Benzonase, US Pat. No. 5,173,418), the nuclease-treated virus is concentrated (e.g., by use of ultrafiltration). using a filter having a molecular weight cutoff of, e.g., 500 kDa), and the concentrated virus is formulated for vaccination purposes. Details of this method are provided in WO 03/060088 A2, which is incorporated herein by reference. In addition, methods for producing chimeric viruses are described in the documents cited above in reference to the construction of chimeric virus constructs. Formulation of the chimeric viruses used in the methods of the invention can be carried out using methods that are standard in the art. Numerous pharmaceutically acceptable solutions for use in vaccine preparation are well known and easily can be adapted for use in the present invention by those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences (18th edition), A. Gennaro editor, 1990, Mack Publishing Co., Easton , Pennsylvania, United States). In two specific examples, viruses are formulated in Earle's Minimum Salt Essential Medium (MEME) containing 7.5% lactose and 2.5% human serum albumin or MEME containing 10% sorbitol. However, chimeric flaviviruses can be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline. In another example, viruses can be administered and formulated, for example, in the same manner as the 17D yellow fever vaccine, e.g., as a clarified suspension of infected chicken embryo tissue or a fluid harvested from cultures. cell phones infected with a chimeric virus. The chimeric flavivirus vaccines of the invention are stored in a conventional manner either in the form of a frozen liquid composition or in the form of a lyophilized product. For that purpose, the chimeric flaviviruses can be mixed with a diluent in a conventional manner a buffered aqueous solution comprising cryoprotective compounds such as sugar alcohol and stabilizer. Before use, the lyophilized product is mixed with a pharmaceutically acceptable diluent or excipient such as a sterile solution of 4% NaCl to reconstitute a liquid injectable chimeric flavivirus vaccine.

In the method of the invention, the chimeric flavivirus vaccine can be a monovalent, bivalent, trivalent or tetravalent vaccine. According to one embodiment, the chimeric flavivirus vaccine is a monovalent vaccine in which the chimeric virus is composed of a skeleton of YF17D-204 in which the sequences encoding the pre-membrane and capsule proteins of the virus of yellow fever, the pre-membrane and capsule proteins of dengue 2 PUO-218 virus are replaced. According to another embodiment, the chimeric flavivirus vaccine is a tetravalent vaccine, that is, a vaccine comprising chimeric viruses expressing antigens of the four serotypes of dengue virus (1 to 4). In a particular embodiment, this tetravalent vaccine advantageously includes four chimeric flaviviruses composed respectively of a YF17D-204 backbone in which the sequences encoding pre-membrane and yellow fever virus capsule proteins are replaced with sequences encoding the proteins of pre-membrane and capsule of dengue virus 1 PUO-359 (YF / dengue 1), dengue virus 2 PUO-218 (YF / dengue 2), dengue virus 3 PaH-881/88 (YF / dengue 3) , or dengue virus 4 1228 (YF / dengue 4). This specific tetravalent vaccine is named in Example 2 following ChimeriVax-DEN tetravalent. Examples of tetravalent chimeric flavivirus vaccines Suitable for use in the method of the invention are also described in detail in WO 03/101397, the content of which is incorporated herein by reference. Multivalent vaccines can be obtained by combining single monovalent dengue vaccines. The chimeric viruses of the invention can be administered using methods that are well known in the art. For example, viruses can be formulated as sterile aqueous solutions containing between 102 and 107, e.g., containing between 103 and 106, such as 104, 105, or 106 infectious units (e.g., plaque forming units or infectious doses in tissue culture) by serotype in a dose volume of 0.1 to 1.0 mL, to be administered by, for example, subcutaneous, intramuscular, or intradermal routes. In one embodiment, the chimeric flavivirus vaccine is a monovalent, bivalent, trivalent, or tetravalent vaccine comprising advantageously 105 pfu per serotype, per dose, and it is administered subcutaneously. In addition, because flaviviruses may be able to infect a human host through mucosal pathways, such as the oral route (Gresikova et al., "Tick-borne Encephalitis" in The Arboviruses, Ecology and Epidemiology, Monath (editor), CRC Press, Boca Raton, Florida, United States, 1988, volume IV, 177-203), an administration by mucosal routes (e.g., oral) could also be contemplated. Optionally, adjuvants that are known to the Those skilled in the art can be used in the administration of the viruses used in the invention. Adjuvants that can be used to enhance the immunogenicity of chimeric flaviviruses include, for example, Toll-like receptor agonists and antagonists (TLRs). Immunization Methods As mentioned above, the invention generally involves administration of a yellow fever vaccine strain (e.g., a strain YF17D, as mentioned above), followed by administration of one or more chimeric flaviviruses, in each of which the pre-membrane and capsule proteins of the yellow fever virus have been replaced with the corresponding proteins of a dengue virus (serotype 1, 2, 3, or 4). The yellow fever virus vaccine is administered using standard methods (e.g., by subcutaneous, intramuscular, or intradermal injection, or by percutaneous administration using a device that delivers viruses to the superficial skin), in varying amounts of, for example. , 2-5 (e.g., 3 or 4) log10 plaque forming units (PFU) per dose, which is typically a volume of about 0.5 mL for subcutaneous injection, 0.1 mL for intradermal injection, or 0.002-0.02 mL for percutaneous administration. To allow sufficient time for rest of the innate immune response induced by the yellow fever vaccine, the chimeric flavivirus vaccine is administered at less between 30 days and 10 years, in particular between 30 days and 5 years, such as between 30 days and 1 to 3 years, advantageously, 30, 60, or 90 days, after the yellow fever vaccine, using standard methods and in amounts ranging from 102 and 107, e.g., of 103 and 106, such as 104, 105, or 106 infectious units (expressed as pfu or infection dose in tissue culture) by serotype per dose. In addition, in the case of administration of bi-, tri-, or tetra-valent formulations (see below), in general, the amounts of each chimera in such a vaccine are equivalent, although the use of different amounts of each chimera also it is included in the invention. The methods of the invention may thus involve, for example, administration of a yellow fever virus vaccine on Day 0 and administration of a chimera YF / dengue-1, YF / dengue-2, YF / dengue-3, and / or YF / dengue-4 on Day 30 (or at a later time, as mentioned above). The chimera can be administered as a monovalent vaccine (ie, a vaccine including only one of the following chimeric viruses: YF / dengue-1, YF / dengue-2, YF / dengue-3, or YF / dengue-4), a bivalent formulation (e.g., a vaccine including two of the chimeras listed above, e.g., advantageously including YF / dengue-2 and YF / dengue-4, or YF / dengue-2 and YF / dengue-3) , or YF / dengue-2 and YF / dengue-1), a trivalent vaccine (eg, a vaccine including three of the chimeras listed above, advantageously, a vaccine comprising YF / dengue-2, YF / dengue- 1, and YF / dengue-4, or YF / dengue-2, YF / dengue-3, and YF / dengue-4), or a tetravalent vaccine. The method of the invention leads to a seroconversion (ie, induction of a neutralizing immune response) for four dengue serotypes after only one dose of the chimeric flavivirus vaccine. Although additional doses of the chimeric flavivirus vaccine are not needed to achieve the desired seroconversion and long-term cross-neutralization immune response, administration of booster doses of the chimeric flavivirus vaccine is contemplated in the present invention. Booster doses of the chimeric vaccine of the invention may be necessary to sustain the immune response of cross-neutralization for a longer period of time and may be administered between 6 months and 5 to 10 years after the first dose of dengue vaccine. chimeric, eg, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years, or even 10 years after the first dose of chimeric flavivirus vaccine. The chimeric flavivirus booster vaccine may be different or advantageously identical to the first chimeric flavivirus vaccine administered. The description given above with respect to the chimeric flavivirus vaccine to be administered in the method of the invention applies mutatis mutandis to the chimeric flavivirus booster vaccine. The reinforcement can thus be a monovalent vaccine, bivalent, trivalent, or tetravalent, with respect to the serotypes of dengue present in the vaccine. So, a eg a method of the invention may involve administration of a dose of yellow fever vaccine, followed by a dose of a monovalent chimeric flavivirus vaccine (dengue 1, 2, 3, or 4, advantageously, dengue 2), which is then followed by administration of (i) a monovalent chimeric flavivirus vaccine of the same or different serotype as the chimaera initially administered (advantageously of serotype 4), (ii) a bivalent chimeric flavivirus vaccine, which may or may not include the same serotype as the initial chimera (v.gr., advantageously dengue 1 and 2 followed by dengue 3 and 4), (iii) a trivalent chimeric flavivirus vaccine, which may or may not include the same serotype as the initial chimera, or (iv) a tetravalent chimeric flavivirus vaccine. The chimeric booster flavivirus vaccine is advantageously identical to the first chimeric flavivirus vaccine with respect to its antigen composition. The invention thus also relates to a composition for inducing in a patient a long-term, cross-neutral immune response to the dengue virus including (i) a yellow fever virus vaccine and (ii) a chimeric flavivirus vaccine for a sequential administration, in which the chimeric yellow fever vaccine is administered at least 30 days and up to 10 years after administration of the yellow fever virus vaccine. The invention also includes kits that include a yellow fever virus vaccine and / or one or more chimeric flavivirus vaccines, as described herein. Kits of the invention may also include instructions for using the kits in the vaccination methods described herein. These instructions may include, for example, indications about the quantities of vaccine to be administered and / or information on when the vaccines should be administered. All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention is based, in part, on the experimental results described in the following examples. EXAMPLES In the examples set forth below, experiments and clinical studies are described which show the effects of prior immunity to yellow fever virus in subsequent vaccination with a chimeric YF / dengue-2 (example 1) or a tetravalent vaccine (YF). / dengue-1, YF / dengue-2, YF / dengue-3, and YF / dengue-4) (example 2). These studies include neutralizing antibody assays, seroconversion, viremia, and T cell responses. Example 1: ChimeriVax-Den2 Commercial YF17D vaccine (YF-VAX) was purchased from Aventis- Pasteur, Swiftwater, Pennsylvania, United States. ChimeriVax-Den2 is a genetically engineered, attenuated, live virus, in which the sequences encoding two structural proteins (prM and E) of the YF17D vaccine virus are replaced with the corresponding sequences of the DEN2 virus (strain PUO-218 isolated from a case of classical dengue fever, Bangkok, Thailand). The genetic construction of a chimeric viral genome is achieved using circular cloned deoxyribonucleic acid (cDNA). Full-length cDNA is transcribed to ribonucleic acid (RNA) and the RNA is used to transfect cell cultures, which produce live virus (Guirakhoo et al, J "Virol 75: 7290-7304, 2001). was produced according to Good Manufacturing Practice (cGMP) The virus was grown in Vero cells (kidney of African green monkey) from cell banks that have been tested for adventitious agents, according to the guidelines of the Administration of Food and Drug Administration (FDA) for products derived from mammalian cell cultures Supernatant fluid from cultures of Vero cells containing vaccine viruses are harvested, clarified from cellular waste by filtration, and are treated with a nuclease (Benzonase) to digest nucleic acid molecules derived from host cells.Vulum in volume treated with nuclease is then concentrated by ultrafilt ration and purified by diafiltration The vaccine is formulated with Human Serum Albumin (HSA) USP (2.5%) and lactose USP (7.5%). The vaccine was shown to be sterile and free of mycoplasma, retroviruses [by Transcrip-Increased Reverse Rate by Product (PERT)], and adventitious viruses by in vitro and in vivo tests. The final vaccine bottle is tested for sterility, potency, identity, pH, appearance, osmolarity, HSA, lactose, endotoxin, safety (general safety modified in mice and piglets of the Indies), and neurovirulence in mice. Preclinical studies in monkeys showed that ChimeriVax-DEN2 is highly immunogenic and well tolerated after dose inoculation varying from 2 to 5 log10 PFU (Guirakhoo et al, J ", Virol 74 (12): 5477-5485, 2000). Low-grade viremia occurred during the first week after vaccination in monkeys, similar to that induced by 17D yellow fever vaccine.A single subcutaneous injection of 2 log10 PFU vaccine (the minimum tested dose) induced neutralizing antibodies after 15-30 days, which protected against challenge with wild type DEN2 virus Clinical study with monovalent ChimeriVax-DEN2 vaccine A single-center, double-blind, randomized outpatient study was carried out After selecting, susceptible subjects Yellow fever (YF) eligible were randomized to a single vaccination with a low or high dose of ChimeriVax-DEN2 (3.0 or 5.0 log10 plaque forming units) or YF-VAX. There was also an open component in which the antibody response to high-dose ChimeriVax-DEN2 vaccination was evaluated in subjects immune to YF. Subjects were followed on Days 1-11, 21, and 31 for antibody response and safety evaluations, and the durability of the antibody response was evaluated at 6 and 12 months post-vaccination. After selection, 42 subjects susceptible to YF eligible were randomized equivalently into 3 groups (high or low dose of ChimeriVax-DEN2 or YF-VAX). On Day 1, 14 subjects received a single subcutaneous (SC) vaccination with ChimeriVax-DEN2 (high or low dose) or YF-VAX. 14 additional subjects who were immune to YF (from vaccination against YF previously done 6 months to 5 years before administration of chimeric dengue vaccine) received ChimeriVax-DEN2 high dose. Subjects returned to the clinic on Days 2-11, 21, and 31. Safety assessments were conducted at specific time points during Days 1-31. Antibody responses to homologous vaccine strains and wild-type strains of DEN2, and neutralizing antibodies to YF17D and prototype strains of DEN 1-4, were measured on Days 1 (pre-vaccination) and 31. The study was the view after the end of the treatment period, and the subjects were evaluated in 6 and 12 months post-vaccination for the durability of antibody response. The proportion of subjects who developed neutralizing antibodies at a level > 1:10 to different strains represent- The four dengue serotypes were determined. The effect of previous immunization with YF on the seroconversion rate of DEN2 in the immune groups to YF and susceptible to YF receiving high dose of ChimeriVax-DEN2 was analyzed. The geometric mean of neutralizing antibody titers in each treatment group and for all four dengue serotypes was measured at various time intervals after vaccination, up to 12 months. Viremia Virus circulating in the blood (viremia) is a measure of replication of the different attenuated, live vaccines used in the study. The viremia was tested by a plaque assay on Vero cells. The number of subjects who developed viremia in the 11 days after vaccination is shown by the day of the visit in Table 1. More subjects susceptible to YF vaccinated with ChimeriVax-DEN2 than YF-VAX developed viremia in one or more days of study : 8 (57%) in the group ChimeriVax-DEN2 log10 5.0 PFU and 9 (64%) in the group ChimeriVax-DEN2 log10 3.0 PFU, compared with 2 (14%) in the YF-VAX group. Slightly larger numbers of subjects immune to YF, compared to subjects susceptible to YF, developed viremia after vaccination with ChimeriVax-DEN2 5.0 log10 PFU (11/14 [79%] in subjects immune to YF vs. 8/14 [57%] in subjects susceptible to YF), but the difference was not statistically significant (p = 0.4724). Most subjects developed viremia between days 5-7. Quantitative viraemia measurements (mean peak, average duration, AUC) also were greater in the immune group to YF, but again the differences were not statistically significant and importantly no impact on the safety profile was observed. Table 1. Summary of viremia measures UFP plaque forming units measured in Vero cell cultures 1 Garlic area curve r Comparison in pairs of Ch? mepVax-DEN2 5 0 loglO in subjects susceptible to YF against immune Neutralizing antibodies Three different wild type dengue type 2 strains (16681, JAH, and PR-159), as well as the homologous vaccine strain (ChimeriVax-DEN2), were used in neutralization tests. Wild type strains of heterologous serotypes of Dengue 1 (16007), 3 (16562), and 4 (1036) were also used to measure the amplitude of the neutralizing antibody response. The proportion of subjects seroconverting (demonstrating a neutralizing antibody titer at least greater than or equal to 1:10 between Day 1 and Day 30) was determined. In addition, the geometric mean of neutralizing antibody titers was measured. The Plate Reduction Neutralization Test (PRNT50) used for CVD2, PR-159, and JAH comprises the following steps: Serum deactivated with heat was serially diluted twice and mixed with an equivalent volume of virus to achieve 30-50 pfu / well. The serum-virus mixtures were incubated at 4 ° C for 18 +/- 2 hours, then added to Vero cell monolayers in 12-well culture plates. After 60 +/- 10 minutes of incubation, the monolayers were covered with 0.84% carboxymethylcellulose in culture medium. The plates were then incubated at 37 ° C under 5% C02 for 3-5 days. Monolayers were fixed with 7.4% formalin, then blocked and permeabilized with 2.5% dry skim milk in PBS-Tween 20 plus 0.5% Triton X-100. Antidengue primary antibody 2 (3H5, 1: 5,000) was incubated 60 +/- 10 minutes, followed by goat anti-mouse IgG alkaline phosphatase (1: 500). After 60 +/- 10 minutes of incubation, substrate, BCIP-NBT containing 0.36 mM levimasola was added. The reaction stopped after enough staining had occurred. The plates were counted and the PRNT50 titers were counted. The PRNT50 titers are defined as the first dilution of serum in which the plate count is equal to or less than 50% the count of negative control plates. A serum is considered to be positive for the presence of neutralizing antibodies when the neutralizing antibody titer thus determined is at least greater than or equal to 1:10. For the other strains, the PRNT50 trial has been carried performed in another laboratory according to the following protocol described by Russell et al (J. Im unol. 99: 285-290, 1967). The plate count was determined by using the single-layer assay technique of LLC-MK2 plates. Serums are thawed, diluted, and deactivated with heat by incubation at 56 ° C for 30 minutes. Serial dilutions, 4 times, of serum are made (1: 5, 1:10, 1:40, 1: 160, and 1: 640). An equivalent volume of diluted dengue virus to contain about 40-60 pfu is added to each serum dilution tube. Following incubation at 37 ° C for 60 minutes, 0.2 mL are removed from each tube and inoculated onto triplicate wells of confluent LLC-MK2 in a 6-well plate. Each well is incubated at 37 ° C for 90 minutes and the monolayers are thus covered with 4 mL of 1% Carboxi Methyl Cellulose / Modified Earle Medium. The plates are incubated for 7 days at 37 ° C under 5% C02. Plates are then counted, and the PRNT50 is determined by using logarithmic probit paper. The percentage of plate reduction at each dilution level is plotted to determine the 50% reduction title: plate reduction points between 15% and 85% are used. The results are expressed as reciprocal dilution. A serum is considered to be positive for the presence of neutralizing antibodies when the neutralizing antibody titer thus determined is at least greater than or equal to 1:10. Response 30 days after vaccination On Day 31, seroconversion rates were high against type 2 dengue virus in all groups vaccinated with ChimeriVax-DEN2. Low rates of seroconversion to heterologous DEN 1, 3, and 4 serotypes were observed in subjects susceptible to YF inoculated with ChimeriVax-DEN2 at high or low dose. Rates of seroconversion to DEN1 were 23% and 23% in the dose groups of 5.0 and 3.0 log10 PFU, respectively (Table 2); to DEN3 15% and 23%, respectively; and to DEN4 0% and 0%, respectively. In contrast, 100% of YF immune subjects inoculated with ChimeriVax-DEN2 seroconverted all heterologous DEN serotypes. ChimeriVax-DEN2 vaccine induced very low cross-neutralization antibody titers to heterologous serotypes 1, 3, and 4 in subjects susceptible to YF (Table 3). The geometric mean of neutralizing antibody titers against heterologous dengue serotypes on Day 31 was significantly higher in YF immune subjects vaccinated with ChimeriVax-DEN2 than in subjects susceptible to YF. For DEN1, the geometric mean of antibody titers in subjects immune to YF and in subjects susceptible to YF vaccinated with either 5.0 or 3.0 log10 PFU ChimeriVax-DEN2 were 79 against 10 and 12, respectively (p <0.0001). Similarly, for DEN3, titles were 73 against 13 and 12 (p < 0.0001) (Table 3). None of the subjects susceptible to YF seroconverted to DEN4. The geometric mean neutralizing antibody titre to DEN4 in subjects immune to YF was 57.

Table 2 Seroconversion rate (%) by treatment group, day 31 Table 3 Geometric mean of antibody titer by treatment group, day 31 Response 6 months after vaccination The response to 6 months after vaccination is given in Tables 4 and 5, below. At 6 months, seropositivity rates Low to heterologous DEN serotypes 1, 3, and 4 were observed in subjects susceptible to YF inoculated with ChimeriVax-DEN2 at high or low doses. Rates of seroconversion to DEN1 were 23% and 31% in the dose groups of 5.0 and 3.0 log10 PFU, respectively (Table 4); to DEN3 15% and 23%, respectively; and to DEN4 8% and 8%, respectively. In contrast, 100% of the subjects immune to YF inoculated with ChimeriVax-DEN2 were seropositive to DEN1 and 3, and 64% to DEN4. ChimeriVax-DEN2 vaccine induced low cross-reactivity neutralizing antibody titers to heterologous serotypes 1, 3, and 4 in subjects susceptible to YF (Table 5). The geometric mean of titers of neutralizing antibodies against heterologous serotypes of dengue at 6 months were higher in subjects immunized to YF vaccinated with ChimeriVax-DEN2 than in subjects susceptible to YF. For DEN1, the geometric mean of antibody titers in subjects immune to YF and in subjects susceptible to YF vaccinated with either 5.0 or 3.0 loglO PFU of ChimeriVax-DEN2, were 285 against < 10 and 14, respectively. Similarly, for DEN3, titles were 268 against < 10 and < 10 (Table 5).

Table 4 Seropositive proportion (%) by treatment group, 6 months Table 5 Geometric mean of antibody titer by treatment group, 6 months Response 1 year after vaccination In 12 months, seropositivity rates were the highest against type 2 dengue virus in the YF immune group vaccinated with ChimeriVax-DEN2. This was particularly evident when the two strains of DEN2 PR-159 and JAH were considered. These two strains are from the Americas and belong to two distinct variant groups (Americas I and II, respectively). Low rates of seropositivity to heterologous serotypes of DEN 1, 3, and 4 were observed in subjects susceptible to YF inoculated with ChimeriVax-DEN2 at high or low dose. Rates of seroconversion to DEN1 were 23% and 31% in the dose groups of 5.0 and 3.0 log10 PFU, respectively (Table 6); to DEN3 8% and 23%, respectively; and at DEN4 8% and 0%, respectively. In contrast, 100% of subjects immune to YF inoculated with ChimeriVax-DEN2 were seropositive to DEN1 and 3, and 29% to DEN4. ChimeriVax-DEN2 vaccine induced low cross-reactivity neutralizing antibody titers to heterologous serotypes 1, 3, and 4 in subjects susceptible to YF (Table 7). The geometric mean of neutralizing antibody titers against 12-month heterologous heterologous serotypes was significantly higher in YF immune subjects vaccinated with ChimeriVax-DEN2 than in subjects susceptible to YF. For DEN1, the geometric mean of antibody titers in subjects immune to YF and in subjects susceptible to YF vaccinated with either 5.0 or 3.0 loglO UFP of ChimeriVax-DEN2, were 89 against 10 and 13, respectively (p <0.0001). Similarly, for DEN3, titles were 72 against < 10 and < 10 (p < 0.0001) (Table 7).

Table 6. Seropositive proportion (%) by treatment group, 12 months Table 7. Geometric mean of antibody titer by treatment group, 12 months Response of yellow fever antibodies Surprisingly, of the 14 subjects immune to YF who * were inoculated with ChimeriVax-DEN2, only 2 (14%) had a reinforcement in the YF antibody. Thus, while preexisting YF immunity reinforces the response to dengue serotypes 1-4 after vaccination with ChimeriVax, the reciprocal was not true (ie, ChimeriVax-DEN2 did not reinforce antibodies to yellow fever virus). This result is unexpected, given that the mechanism underlying the extended antibody response to dengue in patients immune to yellow fever who received ChimeriVax-DEN2 (shared epitopes between yellow fever and dengue capsule proteins) would have been expected to result in reinforcement in yellow fever antibodies after ChimeriVax-D2. The results illustrate the unpredictability of cross-protection immune responses to flaviviruses and underscore the novelty of the present invention. T cell responses T cell responses were evaluated by production of IFN? in response to viral antigen in culture supernatants. Subjects were selected with inactivated viral cell lysate, which has been shown to generate mainly CD4 + T cell responses to the vaccine, but some CD8 + cells are also produced (Mangada et al., J. Immunol. Methods 284: 89-97, 2004 ). Materials and Experimental Procedures The T-cell response was evaluated on Days 1 and 31 by measuring the production of IFNα. by PBMC stimulated in culture with virus antigen deactivated. White blood cells are collected on Days 1 and 31 in Vacutainer cell preparation tubes (CPT, BDBiosciences) and sent to Acambis, Inc. for isolation and cryopreservation of PBMC. Cells were washed in RPMI 1640, cryopreserved in heat-deactivated human AB serum (SeraCare, Oceanside, California, United States) containing 10% DMSO, stored in liquid nitrogen, and thawed immediately before testing. To measure the production of IFN ?, PBMC were cultured in 96 well flat bottom plates at 1.5 x 10 5 cells per well for 7 days at 37 ° C with 3 virus cell antigens deactivated with glutaraldehyde: (1) ChimeriVax-DEN2 virus (grown on Vero cells), (2) virus strain PU0218 dengue 2 (wild type dengue virus 2 grown on C6 / 36 cells), and (3) YF virus (grown on cells Vero). Controls consisted of infected Vero or C6 / 36 control cells turned off. Disactivated viral antigen or control cell antigen are added at a concentration of 1: 100 (15). PBMC were also stimulated with 1 μg / ml of ConA as a positive control assay. IFNγ production was determined by ELISA using supernatants collected on Day 7. IFNγ ELISA Culture supernatants were analyzed for IFN? by an indirect ELISA assay (Human IFNy Kit OptEIA, BDBiosciences-Pharmingen, catalog number 555142) according to the manufacturer's instructions. Production of IFN cytokine? The cytokine production of IFN? it was compared on Day 1 and Day 31 of the study (before vaccination and on Day 30 after vaccination) by testing the response to virus antigens deactivated. The vaccine administered (Ch? Mer? Vax-DEN2), the wild type dengue virus 2 (PU0218), and the control virus administered (YF-VAX) were tested. Ch? MenVax-DEN2 grown in Vero cells had very low background on Day 0, while dengue 2 virus grown in C6 / 36 cells produced responses in some of the subjects. However, both of these antigens increased the production of IFN? in each of the four vaccine groups. Deactivated YF-VAX was not very immunogenic in any of the subjects, but showed an increase on Day 31 in relation to Day 1, especially in subjects vaccinated with YF. Comparisons between vaccination groups were made using the difference between values on Day 31 and Day 1 (Figure 1) . All the groups responded to each of the deactivated antigens. Subjects who received 103 or 105 PFU of Ch? Mer? Vax-DEN2 vaccine had levels of IFN? equivalents In the IFN ELISA assay, subjects vaccinated with Ch? Mer? Vax-DEN2 had slightly higher responses than subjects vaccinated with YF (not significant) (FIG. 1). Subjects who were pre-immune to YF had an increase in the number of responses and an increase in the level of IFN? medium (figure 1). Table 8 summarizes the results showing the number of answers as a fraction of the total. About 65% of the subjects vaccinated with Ch? MepVax-DEN2 and YF had an IFN? Response. positive to the vaccine administered as a test antigen, while approximately 90% of YF pre-immune subjects vaccinated with Ch? mer? Vax-DEN2 had a positive response (Table 8). Table 8. Number of Subjects that responded to vaccine based on IFN response? Immunization Group CVD2 Dßn2 PU0218 YF CVD2 103 9/14 8/14 2/14 CVD2 105 9/14 7/14 1/14 YF 105 9/14 4/14 3/14 YF-CVD2 105 13/14 7/14 0/14 Positive response defined as 5 times the background, or = 50 pg / ml on day 30 if day 1 is less than 10 pg / ml (below sensitivity) The results of this study show that approximately 65% of the subjects vaccinated with Ch? mer? Vax-DEN2 and YF have a positive T cell response to the vaccine administered as a test antigen, while -90% of pre-immune subjects vaccinated with Ch? mer? Vax-DEN2 had positive responses (defined for an IFN response?). The production of IFN? was the largest in response to the vaccine virus Ch? mer? Vax-DEN2. T-cell responses in this clinical trial were consistent with neutralizing antibody responses, in both doses of stimulated vaccine similar to immune responses of T cells, and immunity prior to fever virus Yellow did not inhibit the response of T cells to ChimeriVax-DEN2. The answers of IFN? were virtually the same for the 2 doses of ChimeriVax-DEN2 (103 and 105 pfu). The response of IFN? a ChimeriVax-DEN2 was not decreased by previous vaccination with yellow fever virus and even larger numbers of responses were observed, suggesting a trend for increased T cell immunity in subjects pre-immune to YF. The deactivated antigen used in the assay identified the strongest responses but did not determine the specific proteins against which the immune response was generated. As long as an inactivated dengue antigen has been used, it is likely that mainly CD4 + responses are measured. Example 2: Tetravalent Dengue Vaccine Design and Study Methods In this example, the immune response to sequential immunization with 17D yellow fever vaccine is evaluated in human subjects followed by administration of a mixture of four (4) chimeric yellow fever viruses each comprising membrane and capsule proteins of dengue serotypes that are different from each other (ChimeriVax-DEN tetravalent). The first stage of the study was to evaluate the safety, tolerance, and immunogenicity of the tetravalent ChimeriVax-DEN vaccine containing serotypes DEN 1, 2, 3, and 4 compared to a yellow fever (YF) vaccine (YF-VAX) and a placebo. The second stage of the test evaluated the safety and immunogenicity of sequential administration of YF-VAX / ChimeriVax-DEN tetravalent against two doses of tetravalent ChimeriVax-DEN given in a range of 5-9 months. The study consisted of a selection period of 3 to 21 days before the first vaccination, a double blind treatment period after the first 1-month vaccination, and a second selection period of 3 to 21 days, before a period of time. of 30-day open label treatment beginning 5 to 9 months after vaccination. A follow-up visit in 12 months was planned. In the first stage of the study, 3 groups of 33 healthy adult male and female subjects received a vaccination of ChimeriVax-DEN tetravalent (group 1), YF-VAX (group 2), or placebo (diluent of YF-VAX -group 3), for a total of 99 subjects. Prior to conducting any procedure related to the study, subjects provided written informed consent. During the selection, eligibility was assessed by a medical history, a physical examination, vital signs, clinical chemistry, hematology and serology (including pregnancy serum in female subjects), and a urine sample for urinalysis. On Day 1, subjects received a double blind subcutaneous vaccination in the deltoid area and then attended the clinic on Days 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 for AE interview and collection of blood sample for viremia. In addition, in Days 5, 9, 11, and 15, subjects provided a blood sample for clinical laboratory evaluations. On Days 11 and 31, and in 5-9 months, the subjects provided a blood sample for antibody analysis. At 5 to 9 months, continuous eligibility was evaluated and an internal medical history was recorded. Eligible subjects received a second vaccination (tetravalent ChimepVax-DEN vaccine) subcutaneously in the deltoid area. Subjects attended the clinic on days 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 afterwards for AE interview and blood sample collection for viremia. Blood samples for antibody tests were obtained 10 and 30 days after this second vaccination. All subjects returned to the clinic 12 months after the initial vaccination (3-7 months after the second vaccination) for antibody tests. The study design is shown in Tables 9 and 10. Table 9. Treatments to be administered on Day 1 Table 10. Treatments to be administered in 5 to 9 months The dose of precise tetravalent ChimeriVax-DEN by serotype is determined by TCID50 assay as being 3 7/3 1/3 8/3 2 TCID50 for serotypes 1, 2, 3, and 4 respectively. The evaluation criteria for immune responses were as follows: follows: The primary end point for immunogenicity is the rate of seroconversion to serotypes 1-4 of dengue on Day 31, using the neutralization test of 50% plate reduction (PRNT50) of serum dilution, to constant virus, carried performed as described in example 1. This analysis defines the seroconversion rates of all four dengue serotypes and of each individual serotype. Subjects who are seronegative in baseline (< 1:10) will require a PRNT50 title of > 1:10 to meet the criteria for seroconversion. Secondary end points included the analysis of the geometric mean neutralizing antibody titer for each serotype of dengue and seroconversion rate 5 to 9 months after the first vaccination and 12 months after the first vaccination (ie, 3-7 months after of the second vaccination, reinforcement). These serological responses are compared for subjects who received (a) a single dose of ChimeriVax-DEN quadrivalent; (b) two doses of tetravalent ChimeriVax-DEN, or (c) one dose of 17D yellow fever vaccine (YF-VAX) followed by 1 dose of tetravalent ChimeriVax-DEN administered 5-9 months later. Results The objective of the study was to evaluate the amplitude of the immune response across all 4 dengue serotypes following different immunization regimens. The goal of immunizing human subjects against dengue virus disease is to achieve a cross-neutralizing antibody response as wide as possible. The immune responses of all subjects (which were susceptible to dengue and yellow fever in baseline) 30 days after the second dose of study drug are shown in Table 11 (results against wild-type strains). 54 subjects (who were susceptible to dengue and yellow fever in baseline) received a single dose of tetravalent ChimeriVax-DEN on Day 1 or placebo on Day 1 followed by a single dose of tetravalent ChimeriVax-DEN in 5-9 months The neutralizing antibody responses 30 days after receipt of the active vaccine in these groups is combined and is shown in the column to the right of Table 11. A minority of the subjects who received a single inoculation of tetravalent ChimeriVax-DEN had a cross-reactive immune response to 3 or 4 dengue serotypes. Only 43% and 17% of subjects who received a dose of tetravalent ChimeriVax-DEN developed neutralizing antibodies to at least 3 or 4 dengue serotypes, respectively. 27 subjects (who were susceptible to dengue and yellow fever in baseline) received 2 doses of ChimeriVax-DEN tetravalent on Day 1 and in 5-9 months (Group 1). The neutralizing antibody responses are shown in Table 11. The amplitude of the neutralizing antibody response in Group 1 was greater than in subjects who received only one dose of tetravalent ChimeriVax-DEN. 55.6% and 40.7% of the subjects receiving two doses of tetravalent ChimeriVax-DEN developed neutralizing antibodies to 3 or 4 dengue serotypes, respectively. 26 subjects (who were susceptible to dengue and yellow fever in baseline) received yellow fever vaccine (YF-VAX) on Day 1 followed by a dose of tetravalent ChimeriVax-DEN in 5-9 months (Group 2). The neutralizing antibody responses in Group 2 are shown in Table 11. The amplitude of the neutralizing antibody response in Group 2 was greater than in subjects who received either 1 dose of ChimeriVax-DEN tetravalent or two doses of ChimeriVax -DEN tetravalent separated by 5-9 months. 92% and 65% of the subjects receiving sequential immunization with yellow fever vaccine and quadrivalent ChimeriVax-DEN vaccine developed neutralizing antibodies to at least 3 or 4 serotypes of the Dengue, respectively. The results clearly show that a sequential immunization regimen in which a yellow fever vaccine is given before a tetravalent ChimeriVax-DEN vaccine results in a superior immune response to dengue with broad cross-reactivity through the dengue serotypes, than You can achieve with one or two doses of tetravalent ChimeriVax-DEN vaccine tested alone. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made to the same without leaving the spirit or scope of the appended claims. All references cited above are incorporated herein by reference.

Table 11. Open Tag Treatment Period Antibody Response Before Vaccination and 10 and 30 Days After Second Vaccination (Administered 5-9 Months After Primary Dosage) Against At Least One Serotype, At Least Three Serotypes and at Four Serotypes of Dengue Strains (types 1-4), by Treatment Group Seropositive Time Group 1 Group 2 Single Dose After [2] ChimeriVax-DEN - »YF '-VAX? Combination Vaccination ChimeriVaX-DEN Ch meriVax-DEN ChimeriVax-DEN [1] (N = 27) (N = 26) (N = 54) At least one serotype 0 days Yes 26 (96 3%) 6 (23 1%) 7 (13 0%) No 1 (3 7%) 20 (76 9%) 47 (87 0%) Missing 0 (0 0% ) 0 (0 0%) 0 (0 0%) 10 days Yes 24 (88 9%) 14 (53 8%) 9 (16 7%) No 1 (3 7%) 10 (38 5%) 43 (79 6%) Missing 2 (7 4%) 2 (7 7%) 2 (3 7%) 30 days Yes 27 (100 0%) 25 (96 2%) 52 (96 3%) No 0 (0 0%) 1 (3 8%) 2 (3 7%) Missing 0 (0 0%) 0 (0 0%) 0 8 (0 0%) At least two serotypes 00 ddííaass Yes 18 (66 7%) 3 (11 5%) 1 (1 9%) No 9 (33 3%) 23 (88 5%) 53 (98 1%) Missing 0 (0 0% ) 0 (0 0%) 0 (0 0%) 10 days Yes 21 (77 8%) 3 (11 5%) 4 (7 4%) No 4 (14 8%) 21 (80 8%) 48 (88 9%) Missing 2 (7 4%) 2 (7 7%) 2 (3 7%) 30 days Yes 23 (85 2%) 24 (92 3%) 42 (77 8%) No 4 (14 8%) 2 (7 7%) 12 (22 2%) Missing 0 (0 0%) 0 (0 0%) 0 (0 0%) At least three serotypes 0 days Yes 8 (29 6%) 1 (3 8%) 0 (0 0%) No 19 (70 4%) 25 (96 2%) 54 (100 0%) Missing 0 (0 0% ) 0 (0 0%) 0 (0 0%) 10 days Yes 15 (55 6%) 2 (7 7%) 1 (1 9%) No 10 (37 0%) 22 (84 6%) 51 (94 4%) Missing 2 (7 4%) 2 (7 7%) 2 (3 7%) 30 days S 15 (55 6%) 24 (92 3%) 23 (42 6%) No 12 (44 4%) 2 (7 7%) 31 (57 4%) Missing 0 (0 0%) 0 (0 0%) 0 (0 0%) All 4 serotypes 0 days Si 4 (14 8%) 0 (0 0%) 0 (0 0%) No 23 (85 2%) 26 (100 0%) 54 (100 0%) Missing 0 (0 0% ) 0 (0 0%) 0 (0 0%) 10 days Yes 7 (25 9%) 1 (3 8%) 0 (0 0%) No 18 (66 7%) 23 (88 5%) 52 (96 3%) Missing 2 (7 4%) 2 (7 7%) 2 (3 7%) 30 days S 11 (40 7%) 17 (65 4%) 9 (16 7%) No 16 (59 3%) 9 (34 6%) 45 (83 3%) Missing 0 (0 0%) 0 (0 0%) 0 (0 0%) [1] Day 0 is the day on which the second vaccination was given in 5- 9 months The antibody measured at this point in time is the result of the first vaccination 5-9 months before [2] Neutralizing antibody titre = 10

Claims (24)

1. CLAIMS 1. A method for inducing a long-lasting, cross-neutral immune response to dengue viruses in a patient, the method comprising administering to the patient: (i) a dose of a yellow fever virus vaccine, and ( ii) a dose of a chimeric flavivirus vaccine comprising at least one chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the capsule protein of the yellow fever virus have been replaced with sequences that encode the capsule protein of a dengue virus, where the chimeric flavivirus vaccine is administered at least 30 days and up to 10 years after administration of the yellow fever vaccine. The method of claim 1, wherein the chimeric flavivirus comprises a yellow fever virus skeleton in which the sequences encoding the membrane and capsid proteins of the yellow fever virus have been replaced with the sequences encoding the proteins of the yellow fever virus. membrane and capsule of a dengue virus. 3. The method of claim 1 or claim 2, wherein the dengue and / or dengue membrane capsule proteins are scrambled proteins. 4. The method of claim 1, wherein the vaccine of chimeric flavivirus is administered to the patient 30, 60, or 90 days after administration of the yellow fever vaccine. The method of claim 1, wherein the chimeric flavivirus is composed of a skeleton of the YF17D virus. The method of claim 1, wherein the yellow fever virus vaccine comprises a vaccine strain YF17D 17D-204, 17D-213, or 17DD. The method of claim 1, wherein a chimeric flavivirus comprises a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with the sequences encoding the proteins of the yellow fever virus. membrane and capsule of a serotype 1 of the dengue virus. The method of claim 1, wherein a chimeric flavivirus comprises a yellow fever virus backbone in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with the sequences encoding the proteins of the yellow fever virus. membrane and capsule of a serotype 2 of the dengue virus. The method of claim 1, wherein a chimeric flavivirus comprises a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with the sequences encoding the proteins of the yellow fever virus. membrane and capsule of a serotype 3 of the dengue virus. The method of claim 1, wherein a chimeric flavivirus comprises a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with the sequences encoding the proteins of the yellow fever virus. membrane and capsule of a serotype 4 of the dengue virus. The method of claim 1, wherein the chimeric flavivirus vaccine is a monovalent vaccine. The method of claim 1, wherein the chimeric flavivirus vaccine is a tetravalent vaccine. The method of claim 1, further comprising administering a booster dose of a chimeric flavivirus vaccine, as defined in (ii) of claim 1/6 months to 10 years after the first dose of the vaccine. Chimeric flavivirus. A kit comprising: (i) a yellow fever virus vaccine, and (ii) a chimeric flavivirus vaccine comprising at least one chimeric flavivirus comprising a yellow fever virus skeleton in which the sequence encoding the yellow fever virus capsule protein have been replaced with the sequence encoding the capsule protein of a dengue virus. 15. The kit of claim 14, wherein the flavivi- The chimeric rus comprises a yellow fever virus skeleton in which the sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with the sequences encoding the membrane and capsule proteins of a dengue virus. 16. The kit of claim 14 or claim 15, wherein the dengue and / or dengue membrane capsule proteins are scrambled proteins. 17. The kit of claim 14, wherein the yellow fever virus vaccine comprises a YF17D strain. 18. The kit of claim 14, wherein the chimeric flavivirus is composed of a YF17D virus backbone. The kit of claim 14, wherein the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the yellow fever virus membrane and capsule proteins have been replaced with the sequences that encode the membrane and capsule proteins of a serotype 1 of the dengue virus. 20. The kit of claim 14, wherein the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the yellow fever virus membrane and capsule proteins have been replaced with the sequences that encode the membrane and capsule proteins of a serotype 2 of the dengue virus. The kit of claim 14, wherein the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the yellow fever virus membrane and capsule proteins have been replaced with the sequences that encode the membrane and capsule proteins of a serotype 3 of the dengue virus. The kit of claim 14, wherein the chimeric flavivirus vaccine comprises a chimeric flavivirus comprising a yellow fever virus skeleton in which the sequences encoding the yellow fever virus membrane and capsule proteins have been replaced with the sequences that encode the membrane and capsule proteins of a serotype 4 of the dengue virus. 23. The kit of claim 14, further comprising at least one booster dose of a chimeric flavivirus vaccine. 24. The use of (i) a dose of a yellow fever virus vaccine, and (ii) a dose of a chimeric flavivirus vaccine comprising at least one chimeric flavivirus comprising a yellow fever virus skeleton in the which sequences encoding the membrane and capsule proteins of the yellow fever virus have been replaced with the sequences encoding the membrane and capsule proteins of a dengue virus, in inducing a long-term cross-neutralization immune response to dengue virus in a patient, where the chimeric flavivirus vaccine is administered at least 30 days and up to 10 years after administration of the vaccine of yellow fever.